RU-2861420-C1 - METHOD FOR OBTAINING COMMON WHEAT LINES WITH ACCELERATED HEADING BY INDUCING MUTATIONS IN PROMOTER REGION OF PPD-1 GENES USING CRISPR/Cas9

Abstract

FIELD: biotechnology. SUBSTANCE: invention relates to the field, in particular to a method for obtaining a common wheat plant Triticum aestivum L. with accelerated heading. Said method involves using a vector construct encoding Cas9 nuclease and containing two RNA guides complementary to regions of the promoter regions of the Ppd-D1 and/or Ppd-B1 genes; adsorbing said construct onto gold microparticles using TransIT-2020 reagent; transferring said mixture into embryogenic calli of common wheat using a biolistic delivery method; regenerating plants and selecting regenerant plants containing nucleotide insertions and/or deletions in the promoter regions of the Ppd-D1 and/or Ppd-B1 genes. EFFECT: method for obtaining a wheat plant with accelerated heading using the CRISPR/Cas9 system, based on inducing targeted mutations in the promoter regions of the PPD-1 genes. 1 cl, 2 dwg, 3 ex

Inventors

• Kiseleva Antonina Andreevna
• Timonova Ekaterina Mikhajlovna
• Berezhnaya Alina Aleksandrovna
• SALINA ELENA ARTEMOVNA

Dates

Publication Date

20260505

Application Date

20250703

Claims (1)

1. A method for producing a Triticum aestivum L. common wheat plant with accelerated heading, comprising: obtaining a vector construct encoding a Cas9 nuclease and containing two guide RNAs complementary to portions of the promoter regions of the Ppd-D1 and/or Ppd-B1 genes; adsorbing said vector construct on gold microparticles using the TransIT-2020 reagent; transferring said mixture into embryogenic calli of common wheat induced from immature embryos using a biolistic delivery method; regenerating plants and selecting regenerated plants containing insertions and/or deletions of nucleotides in the promoter regions of the Ppd-D1 and/or Ppd-B1 genes, wherein the selected plants are characterized by accelerated heading compared to the original genotype.

Description

The invention relates to plant biotechnology and genetic engineering, specifically to a method for producing wheat lines with an edited sequence of the PPD-1 gene promoter region using CRISPR/Cas9 plant genome editing technology. The method utilizes a DNA vector encoding the Cas9 nuclease, a BAR selective marker, and sequences of two guide RNAs. Optimization of heading timing is one of the key tasks in modern breeding practice, given its impact on plant adaptability and productivity. In bread wheat , the Photoperiod-1 ( PPD-1 ) genes play an important role in regulating the duration of the period from germination to heading. The most significant factor promoting early heading in bread wheat is the Ppd-D1a allele, localized on chromosome 2D [Worland AJ et al. The influence of photoperiod genes on the adaptability of European winter wheats // Euphytica. 1998. Vol. 100, No. 1–3. Pp. 385–394]. It is characterized by a large deletion of 2089 bp in the promoter region. It was established that the Ppd-D1a allele induces an early transition to the generative phase, accelerating the onset of heading by 3-5 days even with a long daylight period [Kiseleva AA, Salina EA Genetic Regulation of Common Wheat Heading Time // Russ. J. Genet. Pleiades Publishing, 2018. Vol. 54, No. 4. P. 375-388]. Previously, the development of varieties with desired traits was achieved through directed selection, which utilizes natural genetic diversity. However, in this case, creating a variety with the desired characteristics requires significant time investments [Voytas DF, Gao C. Precision Genome Engineering and Agriculture: Opportunities and Regulatory Challenges // PLoS Biol. 2014. Vol. 12, No. 6. Pp. 1-6]. In recent years, molecular-guided breeding (MGB) methods have become increasingly important, allowing selection by genetic markers and accelerating the breeding process. The MGB principle is based on the identification of markers linked to genes determining important economically valuable traits and the subsequent use of these markers for accelerated plant selection in breeding programs. Various types of DNA markers are used in molecular breeding, including SNPs, SSRs, and allele-specific PCR markers. The use of MOS is particularly effective when the key genes controlling the desired trait are known, as is the case with the PPD-1 genes. Such approaches have already resulted in the development of a number of promising varieties with modified heading dates (patent RU 2710729C1). However, MOS relies on existing natural diversity and does not allow for the creation of fundamentally new alleles. A more promising approach is the use of genome editing technologies, which enable highly precise and efficient targeted modifications to plant DNA, significantly accelerating the development of new varieties with desired characteristics (patent RU 2772578C2). In this context, the present invention proposes an innovative approach to the development of wheat lines with accelerated heading using the CRISPR/Cas9 system, based on the induction of targeted mutations in the promoter regions of the PPD-1 genes. The essence of the invention consists in developing a method for obtaining lines of soft wheat with mutations in the promoter regions of the PPD-1 genes, ensuring a high frequency of occurrence of large deletions/insertions and stable inheritance of target traits . One of the key distinguishing elements of the developed method is the use of two guide RNAs (gRNA18 and gRNA21) that direct Cas9 to two specific sites in the promoter regions of genes.PPD-1, which induces double-strand breaks at two points and significantly increases the likelihood of large deletions or insertions between them compared to using a single guide.To implement the method, the L2_41780_gRNA18+21_Cas9_bar construct was used, containing two previously patented RNA guides (patent RU 2822358C1). Unlike most known approaches based on the direct use of immature embryos as explants, in this study, embryogenic calli induced from immature embryos of Velut ( Triticum aestivum L.) common wheat were used as the starting material. To obtain these calli, immature seeds were collected 14–16 days after anthesis. Isolated caryopses were pre-immersed in a 70% ethanol solution (v/v) for 1 minute, then treated with a 50% household bleach solution (Domestos) for 10 minutes with periodic gentle shaking. After sterilization, the caryopses were rinsed five times with sterile distilled water. Translucent embryos measuring 1–1.5 mm in length were isolated from these calli. 100–150 isolated embryos were placed in Petri dishes, embryonic axis down, scutellum up, in contact with the callus induction medium (WCIM) [Sparks CA, Doherty A. Genetic Transformation of Common Wheat (Triticum aestivum L.) Using Biolistics // Biolistic DNA Delivery in Plants: Methods and Protocols / ed. Rustgi S., Luo H. Springer Science+Business Media, 2020. Vol. 2124. Pp. 229–250]. In our experiment, the WCIM contained 2.5 mg/L